Hepatocellular carcinoma (HCC) ranks sixth in global cancer prevalence and third in cancer-related mortality (1-3). Despite significant advancements in systematic therapies for HCC, including targeted therapy, immune checkpoint inhibitors, and intravenous chemotherapy, the 5-year recurrence rate remains high at 75% (4,5). HCC represents a formidable global public health challenge (6). Recently, genome-wide (7,8) and candidate gene studies (9-12), have revealed several potential diagnostic biomarkers for HCC. Nevertheless, identifying a clinically validated diagnostic marker remains challenging (13).

Aldehyde dehydrogenase 2 (ALDH2) is known for its role in ethanol metabolism, primarily expressed in liver mitochondria where it catalyzes the conversion of toxic acetaldehyde to acetic acid. Numerous studies have investigated associations between ALDH2 genotypes and various human diseases, including coronary artery stenosis (14), myocardial infarction (15), and ischemic stroke (16). Among single nucleotide polymorphisms (SNPs), ALDH2 rs671 is the most extensively studied, incorporating rs60823674, rs4986830, rs4134524, and rs2230021. Individuals carrying at least one variant allele of ALDH2 rs671 demonstrate significantly reduced enzymatic activity (17,18), leading to heightened sensitivity to alcohol, commonly referred to as "Asian Flush Syndrome" (19-21). Moreover, the AA genotype of ALDH2 rs671 has been linked to increased risks of upper digestive tract cancers and head and neck cancers (22,23). Furthermore, ALDH2 polymorphisms influence HCC development in non-alcoholic patients (24,25).

In the literature, several studies have indicated that the ALDH2 rs671 polymorphism does not correlate with HCC susceptibility in East Asian populations (26-29). Nevertheless, the influence of alcohol consumption patterns and ALDH2 rs671 genotypes on HCC development remains uncertain and warrants further investigation. Thus, our study aimed to explore the association between ALDH2 rs671 genotypes and the combined impact of alcohol drinking behaviors with ALDH2 rs671 genotypes on HCC risk in a representative Taiwanese population, comprising 298 HCC cases and 889 non-cancerous healthy controls.

Recruitment of HCC patients and non-cancerous control groups. Patients diagnosed with HCC by expert surgeons at China Medical University Hospital, Taiwan, were eligible for recruitment. HCC patients and non-cancerous healthy subjects who consented to complete a self-administered questionnaire and provided peripheral blood samples were enrolled in the study. Non-cancerous healthy controls, matched for age and sex, were selected from the Health Examination Cohort during the same period. The study design, including exclusion and inclusion criteria, was approved by the Institutional Review Board of China Medical University Hospital (DMR103-IRB-094). Table I summarizes selected information extracted from the questionnaire database of HCC cases and non-cancerous healthy subjects.

Genotyping methodology for ALDH2 rs671 polymorphism. Peripheral blood leukocytes from each participant were isolated using the QIAamp Blood Mini Kit (Blossom, Taipei, Taiwan, ROC), following procedures described in previous publications (30-32). PCR amplification of the ALDH2 rs671 polymorphic site was performed using specific primers: forward 5’-CAA ATT ACA GGG TCA ACT GC-3’ and reverse 5’-CCA CAC TCA CAG TTT TCT CTT-3’, followed by genotyping using PCR-restriction fragment length polymorphism analysis. Genotypic processing was independently conducted by trained researchers (Chang WS, Tsai CW, Wang YC, Shih HY, and Chin YT) in a double-blind manner. All repeated genotyping results were 100% concordant. The physical map illustrating the locations of ALDH2 rs671 is presented in Figure 1.

Statistical methodology. Statistical analyses were conducted using SPSS version 16.0 software (SPSS, Inc., Chicago, IL, USA). The goodness-of-fit chi-square test assessed the Hardy-Weinberg equilibrium in the non-cancerous control group. The differential distributions of age between the HCC case and non-cancerous control groups were evaluated using Student’s t-test. Pearson’s chi-squared test with Yates’ correction examined the differential distribution of various ALDH2 genotypes and alleles. Associations between ALDH2 genotypes and HCC risk, as well as the combined impacts of ALDH2 genotypes with sex or alcoholism, were assessed through multivariable logistic regression analyses, calculating odds ratios (ORs) and corresponding 95% confidence intervals (CIs). A probability value ≤0.05 was considered statistically significant.

The demographic data for the HCC populations. Table I presents the demographic characteristics including age, sex, smoking status, and alcohol consumption habits of the study participants, comprising 298 HCC patients and 889 non-cancerous healthy subjects. Utilizing a matching strategy ensured comparable age and sex distributions between cases and controls (both p>0.05) (Table I). The case group exhibited significantly higher proportions of ever smokers and ever alcohol drinkers compared to the control group (75.2% vs. 65.1% and 69.1% vs. 41.7%, respectively; p=0.0017 and 0.0011) (Table I). These results underscore the role of individual cigarette smoking and alcohol consumption as risk factors for HCC in Taiwan.

Associations of ALDH2 genotypes with HCC risk. Table II displays the distributions of ALDH2 rs671 genotypes among HCC cases and non-cancerous healthy controls. First, genotypic frequencies of the SNP in non-cancerous healthy controls were consistent with Hardy-Weinberg equilibrium (p_HWE=0.8512). Second, ALDH2 rs671 genotype frequencies differed significantly between HCC cases and healthy controls, with higher proportions of the heterozygous variant AG and homozygous variant AA genotypes observed in HCC cases compared to controls (43.6% vs. 41.2% for AG and 12.4% vs. 8.1% for AA genotype, p_trend=0.0311). Logistic regression analyses revealed that the AA genotype was associated with an increased risk of HCC [odds ratio (OR)=1.77, 95% confidence interval (95%CI)=1.38-2.75, p=0.0150] relative to the wild-type GG genotype. In a dominant model analysis, individuals carrying variant genotypes (AG+AA) exhibited a 1.31-fold increased HCC risk (95%CI=1.01-1.71, p=0.0504, Table II).

Contributions of ALDH2 allelic frequencies to HCC risk. Table III presents the allelic frequencies of ALDH2 rs671 among HCC cases and non-cancerous healthy controls. Consistent with the results from Table II, individuals carrying the variant A allele of ALDH2 rs671 exhibited a higher risk of HCC compared to those with the wild-type G allele (OR=1.29, 95%CI=1.06-1.58, p=0.0123, Table III).

Stratified analysis of ALDH2 rs671 genotypes based on sex and alcohol drinking status. We attempted to investigate the combined impact of ALDH2 rs671 genotype with sex and alcohol consumption on HCC risk. Regarding sex, a significant interaction was observed between ALDH2 rs671 genotype and males (p_trend=0.0275), particularly among those carrying the AA genotype (p=0.0145, left part of Table IV). Conversely, no significant interaction was found between genotype and females, whether individuals were AG or AA carriers (p=0.9275 and 0.7003, right part of Table IV). This pattern persisted after adjusting for age, smoking, and alcohol consumption status (Table IV).

Regarding alcohol consumption status, a significant interaction was noted between ALDH2 rs671 genotype and non-drinkers (p_trend=7.9E-8), particularly among those with the AA genotype (p=0.0001, left part of Table V). Conversely, there was no significant interaction between ALDH2 rs671 genotype and drinkers, whether individuals carried AG or AA genotypes (p=0.2163 and 0.7191, right part of Table V). Adjusting for age, sex, and smoking status reaffirmed these findings (Table V).

In the current study, we explored the role of alcohol consuming status and ALDH2 genotype in HCC development. The ALDH2 rs671 genotype is prevalent in East Asian populations, with frequencies ranging from 28-45%, contrasting its rarity in Western populations (33). Some studies suggest that carriers of the variant ALDH2 rs671 genotypes may self-select against alcohol consumption due to the acetaldehyde-induced flushing reaction (20,21). However, recent trends indicate increasing alcohol consumption among East Asian populations (34), potentially contributing to elevated HCC risk (35,36).

Our study highlights that the variant AA genotypes of ALDH2 rs671 are associated with increased HCC risk, particularly among males (Table II, Table III, and Table IV). Furthermore, the ALDH2 rs671 AA genotype significantly enhances HCC risk among non-alcohol drinkers (Table V, left part). These findings contrast with previous reports suggesting inconsistent associations between ALDH2 rs671 polymorphism and HCC risk in alcohol drinkers (37-40).

Of particular interest, our study suggests that the AA genotype of ALDH2 rs671 may lead to severe consequences for alcohol drinkers, potentially resulting in a biased OR estimate (Table V, right part). Conversely, the pronounced effects of the ALDH2 rs671 AA genotype among non-drinkers are evident (Table V, left part), potentially obscuring the inclusion of HCC cases with ALDH2 rs671 AA genotype who were alcohol drinkers and did not survive to be included in our study sample.

In the literature, the ALDH2 rs671 polymorphism has been reported to be associated with HCC risk among patients without alcoholism (24,25). This finding is consistent with our observation that among non-drinkers, carriers of the AA genotype at ALDH2 rs671 had a 7.6-fold increased OR for HCC risk (Table V, left part). However, a meta-analysis indicated that the ALDH2 rs671 polymorphism is not generally associated with HCC susceptibility in East Asian patients with HBV or hepatitis C virus (26). Furthermore, several studies have shown that the ALDH2 rs671 polymorphism is not associated with HCC in East Asian populations (26-29). These discrepancies may stem from differences in study designs that consider virus infections (26,28), or variations in the studied populations such as Japanese, distinct from the Han Chinese population examined in our study (27,29).

This study was conducted in a homogeneous cohort of HCC patients of Han Chinese descent, characterized by a significantly higher frequency of the ALDH2 rs671 "A" allele compared to other populations, as demonstrated in the 1000Genomes Project. Specifically, the ALDH2 rs671 "A" allele frequencies were nearly 0% in African, American, European, and South Asian populations, whereas they were 25.5% in East Asian populations with a sample size of 3,118 subjects (41). Consequently, it is crucial to emphasize that the predictive implications of this study are applicable primarily to East Asian populations. The combined effects of the ALDH2 rs671 genotype with alcohol consumption could not be broadly extrapolated to other populations.

This study provides evidence that variant genotypes at ALDH2 rs671 are associated with increased HCC risk, especially among males. Furthermore, these genotypes significantly elevate HCC risk among non-alcohol drinkers and may have severe consequences for alcohol drinkers. ALDH2 rs671 serves not only as a genetic susceptibility locus for HCC in Taiwanese populations but also as a critical predictor for potential life-threatening outcomes. Individuals carrying the ALDH2 rs671 A-allele, particularly those with the AA genotype, are advised to discontinue alcohol consumption promptly.

All the Authors declare no conflicts of interest regarding this study.

Conceptualization: Chin YT, Wu MH and Bau DT; Data curation: Wu MH, Pei JS, Ke TW and Hung YC; Investigation: Wu MH, Shih HY, Tsai CW, Wang YC and Chang WS; Formal analysis: Pei JS, Chen JC and Chang WS; Writing – original draft: Chang WS and Bau DT; Writing – review & editing: Chin YT, Wu MH, Shih HY, Tsai CW, Pei JS, Ke TW, Wang YC, Hung YC, Chen JC, Bau DT and Chang WS.

The Authors thank Yu-Cheng Luo and Yu-Hsin Yen for technical assistance in DNA extraction, aliquot and storage. This study was supported by grants from Taichung Armed Forces General Hospital (grant number: TCAFGH-D-111020). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.